An electrochemiluminescent sensor and signal tag thereof, method of constructing and detecting aβos

Abstract

The scheme discloses the technical field of biosensors, and particularly relates to a signal label of an electrochemiluminescence sensor, the signal label is Ab2@PdPtB MNPs@luminol signal label, and the signal label comprises PdPtB MNPs, luminol and a labeled antibody Ab2. The SiC@Au-PEDOT NWs are used as a conductive and biocompatible substrate to modify a glassy carbon electrode (GCE) to improve the sensitivity of detection. The core of the technology is that when the AβOs exist, the Ab2@PdPtB MNPs@luminol signal label is combined on the surface of the GCE to realize high-intensity ECL signal output. The developed ECL immunosensor provides a new reliable tool for high-precision analysis of AβOs, provides a new thought and experimental basis for an AβOs clinical test diagnosis method, and provides strong technical support for early diagnosis of AD.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biosensors, and particularly relates to an electrochemiluminescence sensor, a signal tag thereof, and a method for constructing and detecting AβOs. BACKGROUND

[0002] Alzheimer's disease (AD) is a neurodegenerative disease mainly manifested as progressive cognitive dysfunction and behavioral disorders. Recent research reports show that amyloid beta oligomers (AβOs) are the cause of Alzheimer's disease neuron dysfunction and memory impairment, and can be used as a biomarker for diagnosing AD. However, the analysis performance of the existing AβOs detection technology cannot meet the needs of clinical application.

[0003] In recent years, electrochemiluminescence (ECL) as a commonly used biological analysis technology has attracted much attention due to its excellent controllability, wide dynamic response range and high sensitivity. Among numerous electrochemiluminescence reagents, luminol (English: luminol, also known as luminous ammonia. The chemical name is 3-amino-benzene dicarboxylic hydrazine) is widely used in electrochemiluminescence biosensors due to its excellent luminescent performance. In order to obtain high ECL signal intensity, an exogenous co-reactant is needed to stably generate various reactive oxygen species (ROS) (including superoxide (O2 ·- ) and hydroxyl radicals (OH · )), so that the ECL luminescent agent luminol can produce a luminescent signal. Hydrogen peroxide (H2O2) as a commonly used exogenous co-reactant is used to enhance the emission signal of luminol.

[0004] However, the addition of exogenous reactants such as hydrogen peroxide (H2O2) is not only toxic, but also has a long electron transfer path, which can cause measurement errors. SUMMARY

[0005] The present application aims to provide an electrochemiluminescence sensor, a signal tag thereof, and a method for constructing and detecting AβOs based on the deficiencies of the prior art.

[0006] The signal tag of the electrochemiluminescence sensor in the present application is an Ab2@PdPtBMNPs@luminol signal tag, which comprises PdPtB MNPs, luminol and a labeled antibody Ab2.

[0007] The construction method of the PdPtB MNPs is as follows: octadecyl dimethyl ammonium chloride is fully dissolved in ultrapure water, then NH4F solution, H3BO3 solution, H2PdCl4 solution and H2PtCl6 solution are sequentially added, NH3·H2O solution is added after incubation, and stirring is performed until the solution is colorless; then heating is performed to 60-90 DEG C, freshly prepared borane dimethylamine complex solution is injected, and stirring is fully performed; with the addition of the borane dimethylamine complex solution, the solution gradually changes from colorless to dark brown, indicating that the PdPtB MNPs are successfully synthesized.

[0008] The application further provides a construction method of the signal tag of the electrochemiluminescence sensor, wherein the prepared PdPtB MNPs are mixed and stirred with luminol solution at room temperature, then unbound luminol is discarded, and PdPtB MNPs@luminol is formed; then the PdPtB MNPs@luminol is mixed with a labeled antibody Ab2, and after low-temperature (preferably 4 DEG C) stirring and incubation, unbound labeled antibody is discarded, and Ab2@PdPtB MNPs@luminol signal tag is obtained.

[0009] An electrochemiluminescence sensor comprises a modified electrode BSA / Ab1 / SiC@Au-PEDOT NWs / GCE and the Ab2@PdPtB MNPs@luminol signal tag of claim 3.

[0010] The modified electrode is a glassy carbon electrode modified by SiC nanoparticles, 3,4-ethoxydithiophene and HAuCl4 reaction product SiC@Au-PEDOT NWs, the glassy carbon electrode after modification is added with the capture antibody Ab1, then BSA solution is added and incubated, and the BSA / Ab1 / SiC@Au-PEDOT NWs / GCE is obtained.

[0011] Further, after each step in the preparation of the modified electrode is completed, the glassy carbon electrode is gently washed with Tris-HCl (pH 7.4) buffer to remove weakly bound proteins.

[0012] The application further provides a method for detecting AβOs by using the electrochemiluminescence sensor, characterized in that: the AβOs solution is dropped onto the surface of the modified electrode, the signal tag is dropped onto the modified electrode after reaction, and an immune complex is formed after incubation, and the immune complex is placed into a detection system for detection.

[0013] The preparation method of the AβOs solution is as follows: Aβ42 lyophilized peptide is dissolved in a certain concentration of hexafluoroisopropanol, ultrasonic treatment is performed to monomerize Aβ, then hexafluoroisopropanol is evaporated to obtain Aβ, and the obtained Aβ is dissolved in Tris-HCl, and oscillation culture is performed to form AβOs.

[0014] Further, the detection system is detected in PBS (0.1M, pH 7.4), the scanning rate is 0.15V / s, the potential scanning range is-0.2-0.6V, and the photomultiplier voltage is set to 800V.

[0015] The beneficial effects of the present application are: the present technology first uses SiC@Au-PEDOT NWs as a conductive and biocompatible substrate to modify a glassy carbon electrode (GCE) to improve the sensitivity of detection. The results show that when AβOs exist, Ab2@PdPtB MNPs@luminol signal tags are combined on the surface of the GCE, which can realize high-intensity ECL signal output in a neutral working solution without an exogenous co-reactant. The developed ECL immunosensor provides a new reliable tool for high-precision analysis of AβOs, provides a new idea and experimental basis for developing a simple and practical, economical and reliable AβOs clinical test and diagnosis method, and provides strong technical support for early diagnosis of AD.

[0016] Ab2@PdPtB MNPs@luminol signal tags are not only excellent carriers of luminol, but also co-reaction accelerators that can convert dissolved O2 into various reactive oxygen species (ROS) to enhance the ECL signal of luminol. At the same time, we first use high-conductivity SiC@Au-PEDOT NWs as an electrode substrate to improve electronic biocompatibility transfer and obtain satisfactory detection sensitivity. In the presence of AβOs, Ab2@PdPtB MNPs@luminol signal tags are combined on the surface of the modified glassy carbon electrode (GCE), and ECL signals are output in a neutral working solution without an exogenous reactant. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 (A) PdPtB MNPs synthesis process; (B) TEM image of PdPtB MNPs; (C-D) HRTEM image of PdPtB MNPs; (E) HAADF-STEM image of PdPtB@luminol; (F-J) STEM-EDS element analysis image of PdPtB@luminol;

[0018] Figure 2 AβOs detection process;

[0019] Figure 3 XPS spectrum of PdPtB@luminol; (A) N 1s; (B) Pd 3d; (C) Pt 4f; (D) B 1s;

[0020] Figure 4(A) The synthesis process of SiC@Au-PEDOT NWs; (B-C) TEM images of SiC@Au-PEDOT NWs; (D-G) STEM-EDS elemental analysis maps of SiC@Au-PEDOT NWs

[0021] Figure 5 EIS (A) and CV (B) for the step-by-step characterization of the sensor: bare GCE (a), SiC@Au-PEDOT NWs / GCE (b), Ab1 / SiC@Au-PEDOT NWs / GCE (c), BSA / Ab1 / SiC@Au-PEDOT NWs / GCE (d), AβOs / BSA / Ab1 / SiC@Au-PEDOT NWs / GCE (e), PdPtB@luminol-Ab2 / AβOs / BSA / Ab1 / SiC@Au-PEDOT NWs / GCE (f); (C) Detection feasibility of ECL immunosensor: without AβOs (a), with AβOs (b);

[0022] Figure 6 (A) TMB color development reaction images of three mesoporous nanoszymes under the condition without H2O2;

[0023] (a) PdCuB MNPs; (b) PdAgB MNPs; (c) PdPtB MNPs, 2 μl nanoszyme was added into 200 μl TMB solution without H2O2; (B) ECL responses of three mesoporous nanoszymes in (a) 5 mL 0.1 M PBS (pH 7.4) without H2O2; (b) PdAgB MNPs; (c) PdPtB MNPs;

[0024] Figure 7 (A) ECL intensity of bare GCE (a), PdPtB / GCE (b), luminol / GCE (c) and PdPtB@luminol / GCE (d) in air-saturated group, ECL intensity of PdPtB@luminol / GCE (d) in nitrogen-saturated group; OH · (B) and O2 ·- (C) EPR spectra: (a) air-saturated group without PdPtB MNPs, (b) nitrogen-saturated group with PdPtB MNPs, (c) air-saturated group with PdPtB MNPs. (D) Schematic diagram of the mechanism of PdPtB MNPs enhancing luminol ECL signal;

[0025] Figure 8 Mechanism of PdPtB enhancing luminol ECL signal;

[0026] Figure 9TEM images of Aβ peptides after 10 h (A) and 48 h (B) of incubation at 37 °C; (C) ECL response of Aβ Os at different preparation times. Error bars: SD, n = 3;

[0027] Figure 10 Optimization of experimental conditions. (A) Dilution factor of PdPtB@luminol; (B) dilution factor of SiC@Au-PEDOT NWs; (C) pH value of working solution; (D) incubation time of Aβ Os. Error bars: SD, n = 3;

[0028] Figure 11 (A) ECL response of the immunosensor to different concentrations of Aβ Os (a-i: 20 pM, 100 pM, 200 pM, 500 pM, 1 nM, 2 nM, 5 nM, 10 nM and 20 nM); (B) Calibration plot of ECL intensity vs. log of Aβ Os concentration; (C) Specificity of the immunosensor; (D) Reproducibility of the immunosensor (a-c: 100 pM, 500 pM and 5 nM); (E-F) Short-term and long-term stability of the immunosensor (1 nM Aβ Os). Error bars: SD, n = 3. DETAILED DESCRIPTION

[0029] The following is further explained in detail by way of specific embodiments:

[0030] Example 1 Construction of a novel sandwich-type electrochemiluminescence sensor and its application in the detection of Aβ Os.

[0031] 1. Materials and methods

[0032] 1.1 Materials

[0033] Aβ42 lyophilized peptides were purchased from Aladdin (Shanghai, China), anti-Aβ42 antibodies (Ab1, Ab2) were purchased from Merck (Darmstadt, Germany). Octadecyldimethylammonium chloride (DODAC) and chloroauric acid (HAuCl4) were supplied by Alfa Aesar (Ward Hill, USA). Borane dimethylamine complex (DMAB) was purchased from Acros Organics (Belgium). Boric acid (H3BO3) was purchased from Sangon Biotech (Shanghai, China). Ammonium fluoride (NH4F) was purchased from Greagent (Shanghai, China). Palladium chloride (PdCl2), copper nitrate (Cu(NO3)2), potassium tetrachloroaurate (II) (K2PtCl4), acetonitrile and 3,4-ethylenedioxythiophene (EDOT) were purchased from Adamas-beta (Shanghai, China). Bovine serum albumin (BSA, purity > 98%) was purchased from Beyotime Biotechnology (Shanghai, China). Silicon carbide NWs were obtained from Xfnano (Nanjing) Co. Luminol and hexafluoroisopropanol (HFIP) were purchased from Macklin (Shanghai, China). Ammonia solution (NH3-H2O), hydrochloric acid (HC1) and anhydrous ethanol were supplied by Chongqing Chuantong Chemical Co. Ltd (China). All other reagents were of analytical grade and used without further purification. All experiments used Milliq ultrapure water (> 18 MΩ cm -1 , Millipore).

[0034] 1.2 Detection instruments

[0035] ECL measurements were monitored using an MPI-E capillary electrophoresis electrochemiluminescence detector (Remex, Xi’an, China). Electrochemical measurements were performed on a PC-controlled Donghua DH7000 electrochemical workstation (Jiangsu, China). A conventional three-electrode system was adopted: platinum wire (auxiliary electrode), Ag / AgCl electrode (reference electrode) and glassy carbon electrode (GCE, Φ = 3 mm) (working electrode). Transmission electron microscopy (TEM), high-angle annular dark-field (HAADF) and scanning transmission electron microscopy-energy dispersive x-ray spectroscopy (STEM-EDS) were performed using a FEI Talos F200X microscope (USA). X-ray photoelectron spectroscopy (XPS) was collected on an ESCALAB 250Xi spectrometer in the USA. Electron paramagnetic resonance (EPR) was analyzed using a Bruker A300-10 / 12 instrument from Germany.

[0036] 1.3 Synthesis of PdPtB MNPs, as shown in Figure 1 A

[0037] Dissolve 120 mg of DODAC in 40 mL of ultrapure water. Then, add NH4F solution, H3BO3 solution, H2PdCl4 solution and H2PtCl6 solution in sequence. After incubation at 25 °C for 5 min, add NH3-H2O solution (10 wt.%), continue stirring at 25 °C for 5 min until colorless. Heat the mixture to 75 °C in an oil bath, continuously stir for 30 min, then inject 4 mL of freshly prepared 0.1 M borane dimethylamine complex (DMAB) solution into the solution, and stir at 75 °C for 30 min. With the addition of DMAB, the solution gradually changes from colorless to dark brown, indicating the successful synthesis of PdPtB MNPs. Finally, centrifuge the black product, wash it with anhydrous ethanol for 3 times, and disperse it in deionized water.

[0038] 1.4 Synthesis of Ab2@PdPtB MNPs@luminol signal tag

[0039] As shown in Figure 1 A, mix the prepared PdPtB MNPs mesoporous nanoszyme with luminol solution at room temperature, then discard the unbound luminol, to form PdPtB MNPs@luminol. After mixing with Ab2 and incubation at 4 °C, discard the unbound Ab2, collect Ab2@PdPtB MNPs@luminol, and obtain Ab2@PdPtB MNPs@luminol nanotag;

[0040] 1.5 Synthesis of SiC@Au-PEDOT NWs

[0041] Disperse SiC nanoparticles in acetonitrile by ultrasonic wave, then add EDOT and HAuCl4 into the SiC NWs dispersion solution in sequence. Stir at room temperature until the solution gradually changes from yellow to purple black. Finally, centrifuge the solution, wash with acetonitrile, and obtain SiC@Au-PEDOT NWs.

[0042] 1.6 Preparation of AβOs

[0043] Firstly, dissolve Aβ42 lyophilized peptide in hexafluoroisopropanol (HFIP) with a concentration of 1 mg / mL (purity of 99.5%). Ultrasonic treat the solution for 10 min to monomerize Aβ, then evaporate HFIP under the warm flow of high-purity nitrogen. Dissolve the obtained Aβ in 10 mM Tris-HCl (pH 7.4) and incubate at 37 °C with shaking to form AβOs. Aβ monomers aggregate to form AβOs, which are stored at -80 °C for later use.

[0044] 1.7 Construction of sandwich-type electrochemiluminescence sensor

[0045] Prior to measurements, bare GCE was polished with Al2O3 powder for 5 min to obtain a mirror-like appearance. After ultrasonic cleaning with deionized water for three times, the electrode surface was dried with nitrogen for modification. As shown in Figure 2 B, the SiC@Au-PEDOT NWs solution was dropped on the clean GCE surface and dried at 37 °C, and then 10 μL of Ab1 (10 μg / mL) was dropped on the modified electrode surface and incubated at 37 °C for 1 h. Subsequently, 10 μL of 1.0 wt% BSA solution was added on the electrode and incubated at 37 °C for 1 h to block the non-specific adsorption sites. Finally, the constructed ECL immunosensor (BSA / Ab1 / SiC@Au-PEDOT NWs / GCE) was rinsed with 0.02 M Tris-HCl (pH 7.4) and stored at 4 °C for later use.

[0046] 1.8 Electrochemiluminescence detection steps

[0047] 10 μL of different concentrations of AβOs solution was dropped on the BSA / Ab1 / SiC@Au-PEDOT NWs / GCE surface and reacted at 37 °C for 1 h. Subsequently, 10 μL of Ab2@PdPtB MNPs@luminol nanolabels was dropped on the modified electrode and incubated at 37 °C for 1 h to form a sandwich-type immunocomplex for further determination, and the detection process is shown in Figure 2 After each modification step, the electrode was gently rinsed with 0.02 M Tris-HCl (pH 7.4) to remove weakly bound proteins. All ECL measurements were performed in 5 mL PBS (0.1 M, pH 7.4) with a scan rate of 0.15 V / s and a potential scan range of -0.2-0.6 V, and the photomultiplier voltage was set at 800 V.

[0048] Example 2

[0049] Characterization of PdPtB MNPs and SiC@Au-PEDOT NWs

[0050] To verify the successful preparation of PdPtB MNPs, HRTEM, SEM, XPS and STEM-EDS analysis techniques were used to characterize the PdPtB MNPs. The simple synthesis of PdPtB MNPs is shown in Figure 1 As shown in Figure 1 B, the synthesized PdPtB MNPs are highly dispersed spherical nanoparticles with an average diameter of about 100 nm. The HRTEM image further proves that the PdPtB MNPs are dendritic molecular structures with a cluster network Figure 1 C-D). In addition, the nanostructure and elements of PdPtB@luminol nanolabels were studied by high-angle annular dark-field (HAADF)-STEM and STEM-EDS element mapping analysis. As shown inFigure 1 As shown in FIG. 13E, the combination of luminol and PdPtB MNPs did not change the original mesoporous nanocluster structure of the nanoparticles. Meanwhile, STEM-EDS elemental mapping showed that Pd, Pt and B were uniformly distributed in the mesoporous nanospheres, and the N of the luminescent luminol was uniformly distributed on the surface of the nanoparticles Figure 1 Figure 3 As shown in FIG. 14F-J, the composition and chemical bonds of the PdPtB@luminol nanolabels were further analyzed by XPS. The results showed that the synthesis of the PdPtB@luminol nanolabels was successful.

[0051] Figure 4 A shows the synthesis process of SiC@Au-PEDOT NWs. As shown in Figure 4 B-C, the synthesized SiC@Au-PEDOT NWs have a linear structure and a unique nanoporous surface morphology, and the length of the SiC@Au-PEDOT NWs is 6-50 μm, and the width is uniform, about 350 nm. EDS elemental mapping shows the existence and uniform distribution of Si, S and Au in the nanowires Figure 4 D-G), further proving the successful preparation of SiC@Au-PEDOT NWs.

[0052] Example 3 Electrochemical characterization of the ECL immunosensor

[0053] In order to verify the step-by-step modification process of the ECL immunosensor, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the ECL biosensor in a 5 mM [Fe(CN)6] 3- / 4- solution containing 0.1 M KCl.

[0054] In EIS, the diameter of the semicircle represents the electron transfer resistance. As shown in Figure 5 ​As shown in Figure A, the bare GCE exhibits a semi-circular shape (curve a). When SiC@Au-PEDOT NWs are assembled onto the GCE, the EIS of the modified GCE becomes linear (curve b), indicating that SiC@Au-PEDOT NWs can significantly enhance electron transfer between the GCE and the electrolyte solution. This is likely due to the conductive polymer PEDOT transferring charge through electronic and ionic mechanisms, resulting in a significant reduction in impedance. Furthermore, the one-dimensional anisotropic structure and large surface-to-volume ratio of the nanowires provide more electroactive sites. As expected, the electron transfer resistance (Ret) increases significantly as Ab1 is tethered to the modified GCE (curve c), likely due to the hindrance of electron transfer by biomacromolecules. Subsequently, BSA, AβOs, and Ab2@PdPtB MNPs@luminol bioconjugates were layered and assembled onto the modified GCE (curves d, e, and f), and a corresponding increase in Ret values ​​was observed. Furthermore, the redox peak current during the assembly process of the prepared biosensor was measured using CV. Figure 5 B) CV and EIS results indicate that the designed ECL immunosensor was successfully fabricated.

[0055] To demonstrate the feasibility of the ECL immune sensor, we conducted a target induction feasibility experiment. For example... Figure 5 As shown in Figure C, the ECL value (curve b) of the PdPtB nano-signal enhancement particle sandwich immunosensor incubated with AβOs is significantly higher than that of the negative control (curve a), proving that the immunosensor has the ability to detect AβOs and indicating that the ECL immunosensor was successfully constructed.

[0056] Example 4: ECL mechanism of PdPtB nano-signal enhancer

[0057] First, we also conducted a TMB colorimetric assay to investigate the oxidase-like activity of PdPtB MNPs. For example... Figure 6 As shown in Figure A, compared with other nanozymes (PdCuB MNPs and PdAgB MNPs), PdPtB MNPs exhibited a significant color change, indicating stronger oxidase-like activity. Furthermore, we investigated the ECL value of PdPtB MNPs. Figure 6 As shown in Figure B, PdPtB MNPs can significantly improve the ECL efficiency of luminol in a neutral working solution without H2O2.

[0058] Secondly, the ECL behavior of PdPtB nanoparticles for signal enhancement was studied through comparative experiments. For example... Figure 7As shown in Fig. A, no ECL signal was observed on bare GCE and PdPtB MNPs modified GCE (curve a and b), and only a very weak ECL signal was detected on luminol modified electrode (curve c). As expected, the ECL signal was significantly enhanced after PdPtB@luminol modification on the electrode surface (curve d). In comparison with air-saturated condition, a very low ECL signal was observed in nitrogen-saturated neutral PBS group (curve e). This suggests that dissolved oxygen plays an important role in the constructed ECL system.

[0059] The mechanism of the constructed ECL system was further analyzed by electron paramagnetic resonance (EPR) spectroscopy. As shown in Fig. Figure 7 B, no OH · radicals were detected in air-saturated group without PdPtB nano-signal enhancer particles (curve a). Only a weak EPR signal was observed in nitrogen-saturated group (curve b), which might be due to the catalysis of PdPtB MNPs on trace amount of dissolved O2 in solution. In comparison, in air-saturated group, PdPtB catalyzed dissolved O2 to generate OH · radicals in the presence of PdPtB nano-signal enhancer particles. · The EPR signal of DMPO-OH adduct, which was formed by DMPO trapping OH ·- radicals, exhibited a typical EPR signal with an intensity ratio of 1:2:2:1 (curve c). In addition, the EPR spectrum of O2 · radicals was similar to that of OH · radicals (curve d). These results indicated that PdPtB nano-enhancer catalyzed dissolved O2 to generate OH ·- and O2 2 radicals, resulting in the ECL release of luminol. The mechanism of PdPtB enhancing luminol ECL signal is summarized in Fig. Figure 8 , and its schematic diagram is shown in Fig. Figure 7 D

[0060] Example 5 Optimization of experimental conditions

[0061] To determine the optimal analytical performance, a series of experimental conditions were systematically optimized. With the increase of incubation time, AβOs could further assemble into fibrillar materials. Therefore, we optimized the preparation time of AβOs. As shown in Fig. Figure 9 A, after 10 h of incubation, many spherical, uneven AβOs and a small amount of short Aβ fibrils (AβFs) were observed. With the incubation time extended to 48 h, AβOs further aggregated to form a large number of long fibrils with a length of about 500 nm (Fig. Figure 9 B). To verify that the preparation time of AβOs had a significant impact on detection, we further investigated the ECL reaction of AβOs at different preparation times. As shown in Fig.Figure 9 As shown in Figure C, the developed immunosensor exhibits the highest ECL value at 10 h, which is the optimal incubation time for AβOs preparation.

[0062] The concentration of PdPtB@luminol is crucial for the performance and cost control of luminol luminescence systems. For example... Figure 10 As shown in Figure A, the ECL intensity of the ECL biosensor reached its highest level when the synthesized PdPtB@luminol was diluted 4-fold. This is likely because the accumulation of high-concentration PdPtB@luminol affects the emitter conductivity, while low-concentration PdPtB@luminol leads to incomplete binding between the emitter and the electrode surface, thus affecting the ECL signal expression of the sensor. Subsequently, the dilution factor of SiC@Au-PEDOT NWs, which affects electrode conductivity, was optimized. Figure 10 As shown in Figure B, the current value decreased slightly with increasing dilution factor. We selected a 50-fold dilution factor as the optimal concentration for SiC@Au-PEDOT NWs. The pH value of PBS affects the luminescent properties of PdPtB@luminol and the bioactivity of the antigen and antibody. Therefore, a series of PBS solutions with pH values ​​ranging from 5.6 to 8.6 were set as experimental variables. Figure 10 As shown in Figure C, the maximum signal response of the ECL immunosensor was reached at pH 7.4. Finally, the incubation time for AβOs was optimized. Figure 10 As shown in Figure D, the ECL signal gradually increases with the increase of incubation time, reaching its maximum value at 60 min. Therefore, we believe that 60 min is the optimal incubation time for this work.

[0063] Example 6: Analytical performance of ECL immunosensor detection

[0064] Under optimal experimental conditions, different concentrations of AβOs were measured. Figure 11 As shown in Figure A, within the range of 20 pM to 20 nM (a to i), the ECL intensity increases linearly with the AβOs concentration. Figure 11 As shown in the calibration plot of B, the obtained ECL signal response exhibits a good linear relationship with different AβOs concentrations (RB). 2 =0.9984). The linear equation of the standard curve is I = 4908.19lg C - 4619.2 (I is the ECL intensity, C is the AβOs concentration). The limit of detection (LOD) was calculated to be 10 pM based on the three standard deviations of the blank response values. Furthermore, compared with previously reported AβOs detection methods (Table 1), the prepared ECL biosensor exhibits higher sensitivity and a wider linear range, mainly due to the good oxidase-like activity of PdPtB MNPs and the excellent conductivity of SiC@Au-PEDOT NWs.

[0065] To evaluate the specificity of the designed ECL biosensor, different interfering substances, including BSA, AβMs, AβFs and a mixture containing AβOs were added into the sensing system. The concentrations of AβOs and interfering substances were 1 nM and 10 nM, respectively. As shown in Fig. Figure 11 C, there was no significant difference in the detected ECL signal in the presence of BSA and AβMs compared with the blank group. AβFs group showed a certain ECL signal, which might be due to the incomplete conversion of some AβOs to AβFs during the preparation process. In the presence of target protein AβOs, the ECL signal was significantly increased. The results suggest that the ECL immunosensor has good specificity for the detection of AβOs, which may be attributed to the high affinity between antibody-antigen and the good loading capacity of nanomaterials.

[0066] Repeatability and stability are crucial for the clinical application of immunosensors. Therefore, we used the relative standard deviation (RSD) to verify the performance verification of these two aspects. First, under the optimal experimental conditions, different concentrations of AβOs were measured for 6 times in succession, as shown in Fig. Figure 11 D, the RSDs of 3 concentrations of AβOs were 3.62%, 3.14% and 1.71%, respectively, indicating that the constructed ECL immunosensor has good repeatability. Correspondingly, the short-term stability of the ECL immunosensor was evaluated by conducting potential scanning for 1 nM AβOs for 10 cycles in succession. As shown in Fig. Figure 11 E, we observed that the ECL signal did not change significantly during the continuous scanning process, with an RSD of 1.36%, indicating that the prepared ECL immunosensor has satisfactory short-term stability. We further verified the long-term stability of the prepared ECL immunosensor. As shown in Fig. Figure 11 F, compared with the initial ECL intensity, the ECL intensity decreased by 8.25% after storage at 4℃ for 10 days. This suggests that PdPtB MNPs have persistent and stable catalytic activity.

[0067] Table 1 Comparison of different methods for detecting AβOs

[0068]

[0069] Example 7 Recovery experiment

[0070] To evaluate the potential clinical application of the constructed ECL biosensor, a standard recovery experiment was performed. A known concentration of AβOs was added to artificial cerebrospinal fluid and detected by the developed ECL immunosensor. The corresponding AβOs concentration was calculated by the regression equation. The recovery experiment results are shown in Table 2, with a recovery of 95.26% to 103.85% and RSD < 5.13% (n = 3), indicating that the ECL immunosensor has potential application prospects in detecting cerebrospinal fluid AβOs.

[0071] Table 2 Recovery experiment of ECL immunosensor

[0072]

[0073] *Recovery (%) represents the ratio of calculated concentration to actual added AβOs concentration.

[0074] References

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Claims

1. An electrochemiluminescent sensor, characterized by: The modified electrode BSA / Ab1 / SiC@Au-PEDOT NWs / GCE and Ab2@PdPtB MNPs@luminol signal tag; the modified electrode is a glassy carbon electrode modified by SiC nanoparticles, 3, 4-ethoxydioxthiophene and HAuCl4 reaction, and the modified glassy carbon electrode is added with the capture antibody Ab1 and then incubated with BSA solution, and the modified electrode is obtained; The Ab2@PdPtB MNPs@luminol signal tag comprises PdPtB MNPs, luminol and labeled antibody Ab2, wherein the PdPtB MNPs have oxidase-like activity and can catalyze dissolved oxygen to produce reactive oxygen species, thereby enhancing the electrochemical luminescence signal of luminol without external co-reactants; The construction method of the PdPtB MNPs is as follows: the octadecyl dimethyl ammonium chloride is fully dissolved in ultrapure water, then NH4F solution, H3BO3 solution, H2PdCl4 solution and H2PtCl6 solution are sequentially added, NH3·H2O solution is added after incubation, and stirring is performed until the solution is colorless; then heating is performed to 60-90 DEG C, the freshly prepared borane dimethylamine complex solution is injected, and fully stirred, with the addition of the borane dimethylamine complex solution, the solution gradually changes from colorless to dark brown, indicating that the PdPtB MNPs are successfully synthesized; The prepared PdPtB MNPs are mixed with the luminol solution at room temperature, then the unbound luminol is discarded, and the PdPtB MNPs@luminol is formed; then the PdPtB MNPs@luminol is mixed with the labeled antibody Ab2, and after low-temperature stirring and incubation, the unbound labeled antibody is discarded, and the Ab2@PdPtB MNPs@luminol signal tag is obtained.

2. An electrochemiluminescent sensor according to claim 1, characterised in that: After each step in the preparation of the modified electrode is completed, the glassy carbon electrode is gently washed with Tris-HCl buffer solution with pH=7.4 to remove weakly bound proteins.

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